Synthesis, structure, and glutathione peroxidase-like activity of amino acid containing ebselen analogues and diaryl diselenides

Article abstract of DOI:10.1002/chem.201100930

The synthesis of some ebselen analogues and diaryl diselenides, which have amino acid functions as an intramolecularly coordinating group (Se O) has been achieved by the DCC coupling procedure. The reaction of 2,2′- diselanediylbis(5-tert-butylisophthalic acid) or the activated ester tetrakis(2,5-dioxopyrrolidin-1-yl) 2,2′-diselanediylbis(5-tert- butylisophthalate) with different C-protected amino acids (Gly, L-Phe, L-Ala, and L-Trp) afforded the corresponding ebselen analogues. The used precursor diselenides have been found to undergo facile intramolecular cyclization during the amide bond formation reaction. In contrast, the DCC coupling of 2,2′-diselanediyldibenzoic acid with C-protected amino acids (Gly, L/D-Ala and L-Phe) affords the corresponding amide derivatives and not the ebselen analogues. Some of the representative compounds have been structurally characterized by single-crystal X-ray crystallography. The glutathione peroxidase (GPx)-like activities of the ebselen analogues and the diaryl diselenides have been evaluated by using the coupled reductase assay method. Intramolecularly stabilized ebselen analogues show slightly higher maximal velocity (V_max) than ebselen. However, they do not show any GPx-like activity at low GSH concentrations at which ebselen and related diselenides are active. This could be attributed to the peroxide-mediated intramolecular cyclization of the corresponding selenenyl sulfide and diaryl diselenide intermediates generated during the catalytic cycle. Interestingly, the diaryl diselenides with alanine (l,l or d,d) amide moieties showed excellent catalytic efficiency (k_cat/K_M) with low K_M values in comparison to the other compounds. Peroxidase-like activity: Reaction of compound 1 (see figure) with glutathione (GSH) leads to the formation of the corresponding selenenyl sulfide and diaryl diselenide intermediates. The reaction of these intermediates with H₂O₂ leads to the formation of 1 via arylselenenic acid. This process hampers the GPx-like activity at low GSH concentrations.

Full text of DOI:10.1002/chem.201100930

FULL PAPER
DOI: 10.1002/chem.201100930
Synthesis, Structure, and Glutathione Peroxidase-Like Activity of Amino
Acid Containing Ebselen Analogues and Diaryl Diselenides
Karuthapandi Selvakumar,[a] Poonam Shah,[a] Harkesh B. Singh,*[a] and Ray J. Butcher[b]
Dedicated to Manish Kumar Vishnoi
Abstract: The synthesis of some ebse-
len analogues and diaryl diselenides,
which have amino acid functions as an
intramolecularly coordinating group
(Se···O) has been achieved by the DCC
coupling procedure. The reaction of
2,2’-diselanediylbis(5-tert-butylisoph-
thalic acid) or the activated ester tetra-
kis(2,5-dioxopyrrolidin-1-yl) 2,2’-disela-
nediylbis(5-tert- butylisophthalate) with
different C-protected amino acids (Gly,
l-Phe, l-Ala, and l-Trp) afforded the
corresponding ebselen analogues. The
used precursor diselenides have been
found to undergo facile intramolecular
cyclization during the amide bond for-
mation reaction. In contrast, the DCC
coupling of 2,2’-diselanediyldibenzoic
acid with C-protected amino acids
(Gly, l/d-Ala and l-Phe) affords the
corresponding amide derivatives and
not the ebselen analogues. Some of the
representative compounds have been
structurally characterized by single-
crystal X-ray crystallography. The glu-
tathione peroxidase (GPx)-like activi-
ties of the ebselen analogues and the
diaryl diselenides have been evaluated
by using the coupled reductase assay
method. Intramolecularly stabilized eb-
selen analogues show slightly higher
maximal velocity (Vmax) than ebselen.
However, they do not show any GPx-
like activity at low GSH concentrations
at which ebselen and related disele-
nides are active. This could be attribut-
ed to the peroxide-mediated intramo-
lecular cyclization of the corresponding
selenenyl sulfide and diaryl diselenide
intermediates generated during the cat-
alytic cycle. Interestingly, the diaryl dis-
elenides with alanine (l,l or d,d)
amide moieties showed excellent cata-
lytic efficiency (kcat/KM) with low KM
values in comparison to the other com-
pounds.
Keywords: cyclization · ebselen ·
glutathione peroxidase · nucleophil-
ic substitution · selenium
Introduction
enzyme selenosubtilisine,[12] selenopeptides,[13] and seleno-
dendrimers.[14]
Glutathione peroxidase (GPx), a selenoenzyme, plays a cru-
cial role in the detoxification of hydroperoxides in aerobic
living organism.[1] It catalyzes the reduction of hydroperox-
ides by using glutathione (GSH) as a reducing agent. The
redox transformation at the selenium center has been mim-
icked by several organoselenium compounds. The mimics
comprise of ebselen[2] and its analogues,[3] camphor-derived
cyclic selenenamide,[4] benzisoselenazine,[5] diorganyl disele-
nides with intramolecular secondary Se···D (D=N and O)
bonding interactions,[6] cyclic seleninate[7] and selenenate
esters,[8] azaselenonium salts,[9] spridioxyselenuranes,[10] and
seleninic acid anhydride.[11] They also include artificial
Ebselen (1) is a cyclic selenenamide and a classic example
of a glutathione peroxidase mimic.[2] Its mechanism of
action has been studied by using PhSH as a GSH alternative
(Scheme 1).[15] Mechanistic studies reveal that the Se N
ꢀ
bond in ebselen undergoes a facile cleavage reaction with
PhSH to form the corresponding selenenyl sulfide 2. In ad-
dition to the formation of desired intermediates, selenol 3
[a] K. Selvakumar, P. Shah, Prof. H. B. Singh
Department of Chemistry
Indian Institute of Technology Bombay
Powai, Mumbai—400 076 (India)
Fax : (+022)2576-7152
E-mail: chhbsia@chem.iitb.ac.in
[b] Prof. R. J. Butcher
Department of Chemistry, Howard University
Washington DC 20059 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201100930.
Scheme 1. Catalytic reduction of H2O2.
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and selenenic acid 4, the diselenide 5 is also formed. The
presence of strong Se···O intramolecular coordination in 2
makes the selenium more electrophilic and facilitates the
nucleophilic attack of the thiol at the selenium center in 2.
This leads to the undesirable thiol exchange reaction and
hampers the production of selenol 3.[15] This intermediate
contributes to the poor catalytic activity of ebselen in the
benzene thiol assay, however, it shows significant antioxi-
dant activity in the coupled reductase assay in which GSH is
used a co-substrate.[2] Ebselen is currently being evaluated
as an anti-inflammatory drug due to its relatively low cyto-
toxicity in comparison to the other organoselenium com-
pounds.[2]
amino acid groups as ortho donating group is rare. Only the
synthesis, nitric oxide synthase inhibitor and cytokine induc-
er activity of 20 is known in the literature.[17] This attracted
our attention towards the synthesis and studies on the GPx-
like activity of the ebselen analogues and diselenides that
contain amino acid functions. During the course of our
study, Satheeshkumar and Mugesh communicated the syn-
thesis and GPx-like activity of peptides that contain ebselen
analogues and diselenides.[21] It has been demonstrated that
the presence of a valine residue in the peptide enhanced the
antioxidant property by producing the catalytically active se-
lenol intermediate, which is generally not predominant in
the catalytic cycle of ebselen.
In order to improve its catalytic activity, several modifica-
tions have been made on the structure of ebselen. Majority
of the modifications involved replacement of the N-phenyl
ring with different R groups (R=H (6), CH2CH2OH (7),
and CSNH2 (8))[3] and attaching electron-donating or -with-
drawing groups at the N-phenyl ring (compounds 9–12).
Recently we and others have been involved in the synthe-
sis of ebselen analogues 21,[22] 22,[23] 23–25,[24] and 26,[25]
which are stabilized by an intramolecular coordination. It
has been found that analogues 21, 23, and 25, which have an
additional nitro-, amide- or oxazoline-substituent at the 6-
position, show enhanced catalytic activity in comparison to
ebselen. For instance, the GPx-like activity of 21, 25, and 26
are 9, 3 and about 2 times, respectively, greater than that of
ebselen. This conclusion has been arrived purely on the
basis of the initial reaction rates (V0). Based on computa-
tional chemistry, Pearson and Boyd predicated that the sele-
nium center in such ebselen derivatives (e.g., 21) is sterically
hindered by the substituent at the 6-position.[22b] This hin-
drance prevents nucleophilic attack of the thiol at the seleni-
um center at the selenenyl sulfide stage and thus enhances
the catalytic activity.[22b] Bhabak and Mugesh have systemat-
ically studied the effect of an ortho-OMe group at the
second ortho position of the diaryldiselenides 27–29 by de-
tailed kinetic experiments, in situ characterization of the in-
termediates, and computational chemistry.[26] It has been
concluded that the presence of the second ortho-OMe group
in the diaryl dislelenides 27–29 reduces the thiol exchange
at the selenenyl sulfide stage by Se···O intramolecular inter-
action. This enhances their GPx-like activity in comparison
to the parent compound, which does not have an ortho-
OMe group.[26] Very recently, Coulson and Boyd have stud-
ied the effect of electron-donating or -withdrawing groups
on the GP-like activities of compound 27 and related sys-
tems.[27] It has been predicted that the presence of sterically
bulky groups around the selenium hinders the thiol ex-
change and improves the GPx-like activity. However, to the
best of our knowledge, detailed kinetic studies and in situ
77Se NMR spectroscopic characterization of the intermedi-
ates, on ebselen analogues 21, 23, 25, and 26 have not been
attempted.
The synthesis and studies on the biological activities of
ebselen analogues and diselenides that contain amino acid
function have been the topic of current interest. Ebselen an-
alogues with amino acid functions have been known to show
interesting biological activities such as anti-microbial,[16]
nitric oxide synthase (NOS) inhibitor and cytokine inducer
activity.[17] The GPx-like activity of the amino acid deriva-
tives 13–15 has also been reported.[18] A patent application
has indicated that the anti-inflammatory properties of the
ebselen analogues were improved upon introducing the
amino acid derived functional groups.[19]
The main objective of this work is to compare and con-
trast the GPx-like activity of ebselen analogues that contain
an additional amide substituent at 6-position with ebselen
and its related derivatives by using detailed kinetic experi-
ments, in situ 77Se NMR spectroscopic characterization of
the intermediates, and computational studies. In addition,
we highlight the synthetic aspects of ebselen analogues with
an amino acid function at 6-position.
Although, the GPx-like activities of a few diaryl disele-
nides (16–19)[20] that have ortho-amide substituents have
been known, the GPx-like activity of diaryl diselenides with
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Synthesis and Enzymatic Activity of Ebselen Analogues and Diaryl Diselenides
FULL PAPER
Results and Discussion
Synthetic strategy: The most widely used (till date) and pa-
tented synthetic method for ebselen is the acid chloride
method.[28] It involves the reaction of 2,2’-diselanediyldiben-
zoic acid with thionyl chloride to give a 2-(chlorocarbonyl)-
phenyl hypochloroselenoite.[29] Subsequent reaction of the 2-
(chlorocarbonyl)phenyl hypochloroselenoite with aniline
gives ebselen. Engman et al. introduced for the first time a
more convenient synthetic method.[30] It involves the ortho-
lithiation of benzanilide, selenium insertion followed by cyc-
lization by using CuBr2. Fong and Schiesser investigated the
free radical cyclization of 2,2’-diselenobis(benzanilide) with
peroxides.[31] Very recently, Kumar and co-workers have re-
ported the synthesis of ebselen and related derivatives
through a CuI-catalyzed reaction.[32] It was achieved by sele-
Scheme 2. Reagents: i) SOCl2; ii) RNH2.HCl, Et3N, CHCl3; iii) Li2Se2,
DMF; iv) nBuSeLi, THF; v) Br2, CHCl3.
ꢀ
nium insertion across an Ar X bond (X=Cl, Br, and I) of
an aryl halo-amide followed by
a ring closure reaction. The use
of ortho-lithiation reaction and
radical cyclization is found to
have limited applications in
peptide and amino acid chemis-
try, due to the non-selectivity.
Hence, the acid chloride
method has continued to be the
method of choice for the syn-
thesis of ebselen analogues that
contain reactive functionalities.
Lambert and Christiaens used
the SNAr reaction for the syn-
thesis of ebselen analogue 21.[33]
It involves the reaction of 2-
bromo-3-nitrobenzoic acid with
methyl selenolate to give 2-
(methylselanyl)-3-nitrobenzoic
acid. The reaction of 2-(methyl-
selanyl)-3-nitrobenzoic
acid
with SOCl2 and subsequent re-
action with aniline afforded
compound 21. Recently, our
group has succeeded in the syn-
thesis of compounds 23–25 by
using the SNAr reaction.[24]
However, the viability of this
method has not been tested for
the synthesis of amino acid con-
taining ebselen analogues. We
Scheme 3. Reagents: i) NaOH, THF/H2O (1:1), HCl; ii) Gly-OMe·HCl, 1-hydroxybenzotriazole (HOBT),
Et3N, N,N-dicyclohexylcarbodiimide (DCC), THF; iii) Phe-OMe·HCl, HOBT, Et3N, DCC, THF; iv) N-
hydroxy
ACHUTNGTRENsGUN uccinimide (NHS), DCC, THF; v) R’ACHTUNGTREN(NUNG NH2)CHCOOMe·HCl, Et3N, THF; vi) SOCl2; vii) Gly-
OMe·HCl, Et3N, CHCl3.
thought it worthwhile to synthesize such derivatives by
using the SNAr approach.
Method I involves the nucleophilic displacement of bro-
mine in the isophthalic amides 31 and 32 by a selenium nu-
cleophile (Na2Se2). The reaction of Na2Se2 with 31 was sys-
tematically studied. In the solvents THF or DMF, no prod-
uct formation was observed even after 24 h of heating to
reflux. The change of the nucleophile from Na2Se2 to Li2Se2
in DMF afforded a very small quantity (7%) of the expect-
ed ebselen analogue 34. Even prolonged heating to reflux
did not improve the yield and led to the hydrolysis of the
Synthesis: The precursor 2-bromo-5-tert-butyl-isophthalic
acid (30) was prepared by a reported method.[34] Synthesis
of the ebselen analogues with an amino acid moiety was at-
tempted by four different synthetic methods (Schemes 2 and
3).
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H. B. Singh et al.
methoxy group. The yield of the reaction was significantly
small in comparison to the previously reported yields for
compounds 23–25 (71–85%).[24] Moreover, the separation of
34 from the starting material was found to be very difficult
due to the similar polarities of the starting material and the
product. Also, no product formation was observed when
precursor 32 was treated with Li2Se2 under similar reaction
conditions. The significant reduction in the product yield
suggests that the presence of the steric crowd around the
bromine and the polar amino acid function hampers the nu-
cleophilic attack of Na2Se2 or Li2Se2 at the aromatic carbon
atom. In addition, the formation of the presumed intermedi-
ate 33 would be energetically less favorable due to the steric
crowding around selenium atom (see below).
The n-butyl[35] and methoxylmethyl ether[36] groups pres-
ent in unsymmetrical aryl,alkyl selenides undergo facile re-
ductive elimination upon bromination. The resultant prod-
ucts are the alkyl bromide and the arylselenenyl bromide.
Also, it has been known that the resultant arylselenenyl hal-
ides undergo facile intramolecular cyclization reaction with
a reactive functionality present at the ortho position.[37,38]
Hence, we thought, it could be a better approach to get the
desired ebselen analogue 34 (method II, Scheme 2). The re-
action of nBuSeLi with the aryl halides 31 and 32 resulted
in the formation of the unsymmetrical monoselenides 35
and 36, respectively, in 27% yield. The reaction of 35 with
bromine resulted in the forma-
yield (61%). In an attempt to synthesize the l-phenylala-
nine analogue 39 through method III, we obtained com-
pound 40 (31%) in addition to the desired ebselen ana-
logues 39 (10%). Spectral characterization of compound 40
revealed the presence of a covalently bonded dicyclohexyl
moiety. The yield of the ebselen derivative 39 was signifi-
cantly low compared to the yield of 34. This is presumably
due to 1) the sterically bulky l-phenylalanine residue and
2) the formation of side product 40. Hence, we planned to
improve the yield of 39 through a small alteration in the
synthetic method by involving the active ester 41 (method
IIIa). Compound 41 was synthesized from compound 38
(Scheme 3) through DCC coupling.[40] The reaction of the
active ester 41 with a solution of l-phenylalanine methyl
ester hydrochloride in triethylamine afforded ebselen ana-
logue 39 in 42% yield. After visualizing this, we extended
this procedure for the preparation of the derivatives 42 and
43 (35 and 39%, respectively). Also, the reaction of 38 with
thionyl chloride afforded the acid chloride 44, which, upon
treatment with glycine methyl hydrochloride, gave the de-
sired ebselen analogue 34 in 71% yield (method IV). This
observation reveals that the oxidative cleavage of diselenide
bond in 38 before amide coupling improves the yield of the
product.
The plausible mechanism for the formation of compound
40 is given in Scheme 4. It involves the esterification reac-
tion of 34 in quantitative yield.
However, this method was
again found not suitable for the
synthesis of other ebselen de-
rivatives. It posses problems in
the purification of the unsym-
metrical monoselenides, espe-
cially, when the substituents are
l-tryptophan (compound 36)
and l-phenylalanine.[39]
After observing these practi-
cal difficulties in the incorpora-
tion of selenium in the aryl
bromo precursors 31 and 32, we
decided to synthesize the ebse-
len analogues in the reverse
way (method III, Scheme 3). It
involved the incorporation of
selenium followed by the at-
tachment of the amino acid
groups. The required precursor
diselenide 38 was obtained
from 37.[40] Compound 37 was
prepared by using a reported
procedure.[38] The reaction of
diselenide 38 with glycine
methyl ester hydrochloride by
using the DCC coupling proce-
dure afforded the desired gly-
cine derivative 34 in moderate Scheme 4. Possible reaction pathway for the formation of 40.
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Synthesis and Enzymatic Activity of Ebselen Analogues and Diaryl Diselenides
FULL PAPER
tion of compound 38 with HOBT to form the activated
ester. Upon reaction with two equivalents of C-protected l-
phenylalanine, the activated ester gives the corresponding
amide in which only two ester bonds are replaced with
amide bonds. The resultant amide undergoes intramolecular
cyclization to give the selenol. The selenol is known to be
highly reactive and further reacts with DCC to afford the
cyclic selenide 40.
Kersting et al. proposed for the first time the formation of
selenol intermediates in such disproportionation reactions.[23]
The isolation of compound 40 indirectly confirms the forma-
tion of the selenol intermediate in the disproportionation of
the diaryl diselenide having 2,6-disubstitution. The yields of
isolated 39 and 40 are 10 and 31%, respectively. If this reac-
tion follows the reaction path drawn in Scheme 4 alone, it
should give 1:1 ratio of 39 and 40. The observed yields of
the products indicate that there should be some other side
reactions, which are unclear.
Scheme 5. Reagents: i) R’ACHTGNUTRENNUNG
The following points can be arrived from the above ex-
periments: 1) polar and sterically bulky substituents hinder
the nucleophilic displacement (SNAr) of Br in the aryl bro-
mide by Na2Se2/Li2Se2; 2) steric crowding around the seleni-
um center in the diaryl diselenides (e.g., 33) makes them en-
ergetically less favorable and leads to the facile intramolecu-
lar cyclization; 3) DCC coupling of the diaryl diselenide 38
with C-protected amino acids reduces the yield of ebselen
analogues due to side reactions; 4) method IIIa is found to
be the most convenient for the synthesis of ebselen ana-
logues having ortho-amide donor groups; and 5) as far as
the yields of the ebselen analogues are concerned, method
IV is better. However, it requires the use of corrosive
SOCl2.
Having achieved the cyclization reaction of diselenide 38
by using the DCC coupling reaction, we set out to compare
the cyclization reaction with the mono ortho-coordinated
diselenide 2,2’-diselanediyldibenzoic acid (45). Compound
45 was synthesized by using a reported procedure with slight
modification; metallic sodium was used as a reducing agent
instead of Rongalite.[41] The DCC coupling reaction on this
system did not afford any ebselen analogue, instead it af-
forded the corresponding diselenides 20a (S,S or l,l), 20b
(R,R or d,d), 46, and 47 (Scheme 5). This finding corrobo-
rates the observations of Młochowski et al., where they
have used the acid chloride method instead of the DCC cou-
pling reaction for the synthesis of 20a.[17] This observation is
also consistent with that of Liu et al., where they synthe-
sized organoselenium-bridged bis(b-cyclodextrin)s from 45
through a DCC coupling reaction.[42] Even the active ester
48 (synthesized from compound 45 by using DCC coupling)
was found to be unreactive towards the amino acids under
similar reaction conditions and only the starting material
was recovered from each reaction. The ebselen analogues 50
and 51 could only be obtained by the routine acid chloride
method (through intermediate 49), which involved the thio-
nyl chloride reaction. This contrasting reactivity is essential-
ly due to the presence of a aromatic ring strain in 2,6-disub-
stituted aryl selenium systems.[25b,38] The strain is resulting
THF; ii) NHS, DCC, THF; iii) excess aniline; iv) SOCl2; v) R’-
AHCTUNGTRENN(GNU NH2)CHCOOMe·HCl, Et3N, CHCl3.
from the intramolecular coordination and steric crowding
around the selenium center.[38]
Glutathione peroxidase-like activity: The glutathione perox-
idase-like activity of some of these compounds was studied
by both the coupled reductase and the thiophenol assay
method. The results obtained from the coupled reductase
assay are given in Table 1. The initial reaction rates for the
ebselen derivatives 39, 43, and 51, which contain aromatic
side chains, could not be obtained due to their poor solubili-
ty in water. The ebselen analogues/diselenides with glycine
or alanine methyl ester function are highly soluble in com-
parison to other ebselen analogues at the same concentra-
tion. No precipitation or turbidity was observed in the
buffer system. To understand the effect of the GSH concen-
tration on the reaction rate, the initial reaction rates were
measured at different concentrations of GSH. The plot of
reciprocal of the initial reaction rate versus reciprocal of the
Table 1. Catalytic parameters [maximum velocities (Vmax), Michaelis con-
stants (KM), catalytic constants (kcat), and catalytic efficiencies (h)] for
the reduction of H2O2 by GSH in the presence of ebselen analogues and
diselenides.[a]
Vmax [mmminꢀ1
]
KM [mm]
kcat [minꢀ1
]
h [mꢀ1 minꢀ1
]
1
(25.97ꢁ0.72)
(0.31ꢁ0.09)
(1.30ꢁ0.04) (4.49ꢁ1.19)ꢁ103
20a (35.64ꢁ2.61)
(2.50ꢁ0.75)ꢁ10ꢀ2 (1.79ꢁ0.13) (7.44ꢁ1.51)ꢁ104
(2.30ꢁ0.26)ꢁ10ꢀ2 (1.99ꢁ0.09) (8.72ꢁ0.75)ꢁ104
20b (39.66ꢁ1.51)
34
42
46
(53.85ꢁ4.69)
(34.54ꢁ0.58)
(35.10ꢁ0.45)
(1.41ꢁ0.24)
(0.52ꢁ0.03)
(0.21ꢁ0.01)
(2.47ꢁ0.16) (1.80ꢁ0.37)ꢁ103
(1.73ꢁ0.03) (3.36ꢁ0.15)ꢁ103
(1.76ꢁ0.02) (8.23ꢁ0.42)ꢁ103
[a] The reactions were carried out in phosphate buffer (100 mm, pH 7.5)
at (25ꢁ1)8C, with ethylenediamine tetraacetic acid (EDTA) (1.0 mm),
reduced nicotinamide adenine dinucliotide phosphate (NADPH)
(0.23 mm), H2O2 (0.26 mm), GSH (0.5–2.5 mm for ebselen (1), 34, 42, and
46; 0.025–0.5 mm for 20a and 20b), and one of the catalysts (20 mm). All
experiments were conducted in triplicates.
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H. B. Singh et al.
GSH concentration gave the corresponding catalytic param-
eters for these compounds (ebselen (1), 20a, 20b, 34, 42,
and 46).
Computational studies on thiol exchange and intramolecular
cyclization: The most likely explanation for the negligible
reaction rates (at low GSH concentration) observed for the
catalysts 34 and 42 in comparison to the other catalysts (eb-
selen, 20a, and 46), could be obtained by considering the
structural features of the intermediates. The commonly pro-
posed/observed intermediates in the GPx-like catalytic cycle
of ebselen are: 1) arylselenenyl sulfide 2,[15] 2) selenol 3,
3) selenenic acid 4,[43] and 4) diselenide 5[7a,15] (Scheme 1).
The diselenides that contain secondary amides are also
proven to produce the analogous intermediates.[20] Most
likely, from the available information, the mechanistic as-
pects of ebselen/its analogues and diselenides that contain
secondary amides are similar.[20] The key intermediates, aryl-
selenenyl sulfides and arylselenenic acids, proposed in the
catalytic cycle of the ebselen analogues 34 and 42 as well as
the diselenides 20a and 46, have been modeled. The model
compounds (52–55, 34a/34b, and 46a/46b) are given below.
The ebselen analogues 34 and 42, which have an amide
function at 6-position, showed a slightly higher maximal ve-
locity than ebselen. The Vmax of 34 is nearly double in com-
parison to the Vmax of ebselen. It corroborates with the ob-
servation of Parnham et al.[22a] and our observations.[24] The
diselenides 20a, 20b, and 46 also showed slightly better Vmax
than ebselen. The catalytic efficiency of the diselenides 20a,
20b, and 46 is noticeably higher than that of ebselen and its
analogues 34 and 42. In particular, diselenide 20a, which has
l-alanine residues, shows one order of magnitude higher cat-
alytic efficiency than 46 and other compounds (34 and 42).
To figure out this contrasting behavior of 20a in comparison
to 46, we measured the catalytic parameters of compound
20b, which bears d-alanine residues. It is interesting to note
that the catalytic parameters obtained for compound 20b do
not differ significantly from the catalytic parameter obtained
for 20a. It reveals that the chirality of the alanine does not
play any significant role in altering the catalytic efficiency.
Similar observations were also made for 20a in the thiophe-
nol assay. Its catalytic efficiency is nearly doubled with re-
spect to compound 5 and nearly one order of magnitude
higher than that observed for 46 (see the Supporting Infor-
mation). It was noticed that compounds 20a and 20b satu-
rate at low concentration of GSH (KM ꢂ25 mm). This intuit-
ed us to measure the V0 values for all the compounds at a
concentration of GSH of 25 mm. The results are summarized
in Table 2. The V0 values obtained for 20a and 46 are 3.5
and 2.4 times higher, respectively, than the V0 observed for
ebselen. To our surprise, the V0 values obtained for the eb-
selen analogues 34 and 42, which have 6-substitution, are
negligible and nearly equal to the background or control re-
action. A similar observation is also made at concentration
of GSH of 50 mm. It is also evident from the relatively high
KM values observed for the ebselen analogues 34and 42
[(1.41ꢁ0.24) and (0.52ꢁ0.03) mm, respectively] in compari-
son to the KM value obtained for ebselen [(0.31ꢁ0.09) mm].
Our kinetic experiments support the fact that the affinity of
34 and 42 towards GSH is poor and a high concentration of
GSH is required for the better GPx-like catalytic perfor-
mance.
Their geometries have been optimized by using DFT/
B3LYP/6-31g(d) calculations.[38,45] The results obtained from
the computational calculations are summarized in Table 3.
If we consider the strength of the Se···O interaction in the
selenenyl sulfide intermediates, the selenenyl sulfides that
have 2,6-diamide substitutions are only weakly stabilized
compared to the mono ortho-coordinated selenenyl sulfides.
ꢀ
This is supported by the Se···O bond lengths and O···Se S
Table 3. Aromatic ring strain in the 2,6-diamide substituted selenenyl sul-
fides and selenenic acids.
Table 2. V0 values, measured at low GSH concentrations.[a]
ꢀ
Average torsion
O···Se [ꢂ]
O···Se X [8]
V0 [mmminꢀ1
]
V0 [mmminꢀ1
]
angle [8][a]
[b]
[c]
control
1
20a
34
42
46
(0.63ꢁ0.01)
(6.70ꢁ0.24)
(1.45ꢁ0.32)
34a
34b
46a
46b
52
53
54
55
1.73
3.31
0.40
0.26
1.25
2.25
0.76
0.23
2.928
2.394
2.496
2.383
2.915
2.410
2.499
2.376
147.2
170.2
176.2
172.8
147.6
169.0
175.5
172.7
(12.36ꢁ0.44)
(32.96ꢁ2.21)
(0.96ꢁ0.17)
(1.61ꢁ0.16)
(21.72ꢁ0.73)
(23.58ꢁ0.19)
(0.72ꢁ0.10)
(0.54ꢁ0.03)
(15.86ꢁ0.41)
[a] The reactions were carried out in phosphate buffer (100 mm, pH 7.5)
at (25ꢁ1)8C, with EDTA (1.0 mm), NADPH (0.23 mm), H2O2 (0.26 mm),
and one of the catalyst (20 mm). [b] V0 at c
(GSH)=25 mm. [c] V0 c-
[a] The average torsion angle in an aryl ring is the average of six torsion
angle observed in the aryl ring (sum of six torsion angles divided by six).
ACHTUNGTRENNUNG(GSH)=50 mm. All experiments are conducted in triplicates.
12746
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Synthesis and Enzymatic Activity of Ebselen Analogues and Diaryl Diselenides
FULL PAPER
bond angles. The distance between the oxygen atom and the
selenium atom in 34a is 2.928 ꢂ, which is significantly
longer than the corresponding distance in 46a (2.496 ꢂ).
the cyclic selenenate ester system 56.[38] In situ 77Se NMR
spectroscopy and DFT calculations on compounds 56 and 57
revealed the fact that the selenenyl sulfide intermediate 57
obtained from the cyclic selenenate ester 56 has a significant
aromatic ring strain and undergoes intramolecular cycliza-
tion in the presence of H2O2 to give back 56 though 58. The
basic structures around 34, 42, and 56 are very similar.
Therefore, the very low (negligible) catalytic activity of
compound 34 and 42 at micromolar GSH concentrations
could be attributed to the intramolecular cyclization of the
corresponding selenenic acid obtained from the selenenyl
sulfides. The structural features of compounds 27–29 suggest
that the corresponding intermediate arylselenenic acid do
not have any adjacent reactive function, which will undergo
intramolecular cyclization. In contrary, 34b and 53 have
amide functions, which will undergo intramolecular conden-
sation reactions. Such intramolecular cyclization for the sele-
nenic acid 4 obtained from ebselen (1) is known in the liter-
ature.[15b] Therefore, it may be speculated that the relative
rate of cyclization of selenenic acids 34b/53 and 46b/55
would play a crucial role in defining the catalytic activity.
Recently, Sarma and Mugesh studied the intramolecular
cyclization reaction of arylsulfenic acids 59 and 60.[45] It has
ꢀ
Also, the bond angle O···Se S in 46a (176.28) is more linear
than the angle obtained for 34a (147.28). The weaker O···Se
interaction in 34a is further confirmed from the natural
bond orbital (NBO) analysis.[44] The summary of the NBO
analysis is given in Table 4. The O···Se orbital interaction en-
ergies (EO···Se) obtained for 34a and 52 are much smaller
than those obtained for 46a and 54. This smaller EO···Se
values obtained for the 2,6-diamide-substituted systems are
attributed to the presence of the additional substitution at
the second ortho position, which weakens the Se···O intra-
molecular interaction.[38]
Table 4. Summary of NBO analysis on the selenenyl sulfides.
[a]
[a]
[a]
[b]
qSe
qS
qO
EO···Se
[kcalmolꢀ1
]
34a
46a
52
0.309
0.383
0.285
0.384
0.014
ꢀ0.049
0.012
ꢀ0.048
ꢀ0.623
ꢀ0.639
ꢀ0.626
ꢀ0.634
1.23
16.35
1.27
54
15.88
[a] Atomic charges on the Se/S/O were obtained form natural population
analysis. [b] The orbital interaction energy between the lone pairs of the
ꢀ
*
oxygen atom (nO) and the antibonding orbital of the Se S bond (sSeꢀS
)
was determined by NBO second-order perturbation analysis (sum of the
orbital contributions of two oxygen lone pairs).
The parameter of interest is the natural charge on the se-
lenium and sulfur centers. The natural charges on 34a and
52 are less positive than the natural charges on the selenium
atoms of 46a and 54. The charges on the sulfur atoms in
34a and 52 are more positive than the charges on the sulfur
atoms in 46a and 54. This result suggests that the nucleo-
philic attack of the thiol at the selenium atom (undesired re-
action) is more favorable for 46a and 54. Conversely nucleo-
philic attack at sulfur atom (desired reaction) is more favor-
able for 34a and 52. It corroborates with the observations of
Mugesh and Babhak for the diselenides 27–29.[26] The results
suggest that compounds 34a and 52 partly overcome thiol
exchange with the help of additional substituent at the
second ortho position.
Although compound 34 and 42 partly overcomes the thiol
exchange reaction, they are still inactive at low GSH con-
centrations. This leads us to the question why catalysts 34
and 42 are inactive at low concentrations of GSH? Surpris-
ingly, at the same concentrations ebselen and the diselenides
20a, 20b, and 46 are active. It has been found from the re-
sults presented in Table 3 that the average torsion angles in
the aryl ring of 2,6-diamide-substituted selenenyl sulfides
and selenenic acids (1.25–3.318) are higher than the corre-
sponding angles of mono ortho-amide-substituted systems
(0.23–0.768). The larger torsion angles were found for the
been found that the cyclization reaction of sulfenic acid 59
is extremely slow in comparison to the intramolecular cycli-
zation of 60. The S···N intramolecular coordination in 60
brings the OH group of the sulfenic acid closure to the NH
group of the amide function, thus enhances the rate of its in-
tramolecular cyclization to give the corresponding cyclic sul-
fenamide. The same holds probably true for the selenenic
acid models 34b/53 and 46b/55. Selenenic acids 34b and 53
may undergo more rapid intramolecular cyclization than the
selenenic acids 46b and 55, respectively. This may hamper
the GPx-like activity of the real systems 34 and 42.
To further validate the role of intramolecular cyclization
towards the poor catalytic behavior of the 2,6-substituted
systems 34 and 42 at low GSH concentration, the reaction
profiles for the conversion of 53 and 55 to their correspond-
ing cyclic selenenamides were studied by using DFT calcula-
tions.[46] It has been found that 55 has several energy barriers
for the condensation reaction. The barriers include 1) break-
ing of the intramolecular Se···O secondary bonding interac-
tion, 2) rotation of the amide group, and 3) approach of the
OH group towards the NH group for condensation. In con-
trast, compound 53 is already in the activated state in which
the condensing partners are situated in appropriate positions
and the Se···O interaction lowers the energy barrier for the
ꢀ
ꢀ
C1 C6 and C1 C2 bonds. This leads to a small strain for
2,6-disubstituted aryl selenium compounds. Recently, we
have demonstrated the origin and consequence of the aro-
matic ring strain present in the intermediates obtained from
Chem. Eur. J. 2011, 17, 12741 – 12755
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12747

H. B. Singh et al.
condensation through neighboring group participation. Ad-
ditional features of the reaction profile of 53 are 1) the con-
densation occurs in a single step, 2) the condensation reac-
tion of 53 is exothermic, and 3) the aromatic ring strain
present in 53 is released after the cyclization. These calcula-
tions suggest that the intramolecular cyclization of 53 is
more facile than that of 55.[46]
64.[7a,47] Upon addition of up to three equivalents of GSH,
the peak corresponding to selenol 65 was not observed in
the 77Se NMR spectrum. Attempted the synthesis of the Se-
carboxymethylated derivative 66 was also unsuccessful,[48] it
clearly indicates that the catalytically active selenol (e.g.,
65) is not produced during the catalytic cycle. This is a char-
acteristic catalytic feature of ebselen and its analogues.[21]
This observation is in sharp contrast with our computational
results (natural charge calculations) on 52/34a, which pre-
dicted that such system should overcome the thiol exchange
to produce catalytically active selenol through desired elec-
tronic effects. However, our experimental study suggests
that the additional amide group at the 6-position may steri-
cally block the entry of the second thiol molecule at the se-
lenenyl sulfide stage and prevent the production of the sele-
nol.
Addition of two equivalent of H2O2 to a mixture that con-
tains selenenyl sulfide 63, diselenide 64, and GSH resulted
in the formation of ebselen analogue 62. This result corrobo-
rates our previous observation for the selenenyl sulfide 57,
which underwent a facile intramolecular cyclization to
afford the cyclic selenenate ester 56.[38] The most likely inter-
mediates for this cyclization would be 67 and 68.[38,47] This
provides compelling evidence that selenenyl sulfide 63 and
diselenide 64 undergo intramolecular cyclization in the pres-
ence of H2O2. Engman et al.[47] described that the reaction
of glutathionylated ebselen (selenenyl sulfide) underwent a
slow oxidation reaction with H2O2 to produce selenenic acid
4 and GSSG. This step is considered to be the rate-deter-
mining step of the catalytic cycle of ebselen. The resultant
selenenic acid 4 rapidly reacted with GSH to produce gluta-
thionylated ebselen. Based on
Mechanistic studies by using in situ 77Se NMR spectroscopy:
To gain further insight about the poor catalytic activity of
compounds 34 and 42, we carried out an in situ 77Se NMR
spectroscopic characterization of the intermediates. The re-
action of 34 with thiophenol in CDCl3/CD3OH (2:1) does
not result in the formation of the corresponding selenenyl
sulfide even at very high concentration of PhSH. It might be
due to the poor nucleophilicity of PhSH with respect to
GSH. The reaction of 34 with GSH was not successful due
to the insolubility of GSH in CDCl3/CD3OH (2:1). To over-
come the solubility problem, compound 34 was hydrolyzed
to give the corresponding carboxylic acid 61 (Scheme 6).
The structure of 61 was confirmed with single-crystal X-ray
study. The sodium salt 62 of the carboxylic acid 61, which
was prepared in situ in phosphate buffer under argon atmos-
phere (D2O, pH 7.5), showed a peak at d=915 ppm in its
77Se NMR spectrum. Upon addition of one equivalent of
GSH a peak at d=463 ppm appeared, which corresponds to
the selenenyl sulfide 63. With three equivalents of GSH sig-
nals at d=463 and 440 ppm emerged. The new signal at d=
440 ppm is presumably due to the formation of the corre-
sponding diselenide 64. This could be due to the dispropor-
tionation of selenenyl sulfide 63 to GSSG and diselenide
these results, it is speculated
that the catalytically disadvan-
tageous intramolecular cycliza-
tion of 68 (68!62+H2O) com-
petes with the catalytically ad-
vantageous
glutathionylation
reaction of 68 (68+GSH!63).
Thus, intramolecular cyclization
of 2,6-diamide-substituted aryl-
selenenic acids could be pre-
vented/overcome at high con-
centration of GSH.
In summary, the kinetic ex-
periments, the in situ 77Se NMR
spectroscopy, and the computa-
tional studies provide the fol-
lowing information: 1) the
mechanistic aspect of ebselen
analogues with an amide sub-
stituent at the 6-position is very
similar to that of ebselen; 2) eb-
selen analogues with an amide
substituent at the 6-positions
require high concentrations of
GSH to overcome the intramo-
Scheme 6. Plausible mechanism of the catalytic action of compound 62 at low GSH concentration.
12748
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Chem. Eur. J. 2011, 17, 12741 – 12755

Synthesis and Enzymatic Activity of Ebselen Analogues and Diaryl Diselenides
FULL PAPER
lecular cyclization reaction, which is evident from the KM
values; 3) the selenenic acids that have two ortho amide
functions undergo relatively faster cyclization than the cor-
responding selenenic acids with one ortho amide function.
This is evident from the computational calculations (see
above) and the available literature data on related sulfenic
acids;[45] 5) the slight differences in the catalytic antioxidant
activity of ebselen analogues at high concentration of GSH
may probably arise from the difference in the rate of forma-
tion of the diselenide intermediate and the reactivity of the
corresponding selenenyl sulfide/diselenide intermediates
with H2O2. Pearson and Boyd have suggested that the intro-
duction of an ortho-nitro group to the selenium center of
ebselen may enable the production of selenol by overcoming
the thiol exchange reaction.[22a] In contrast, our experimental
studies on 2,6-diamide-substituted aryl selenium compounds
suggest that such systems do not produce any selenol inter-
mediate in the presence of GSH. The V0 values reported for
compounds 21,[22a] 25,[24] and 26[25] are 9, 3 and about 2 times,
respectively, greater than that of ebselen. The Vmax values of
the amino acid derivatives 34 and 42 are 2.07 and 1.33 times,
respectively, greater than Vmax of ebselen. Among these,
only compound 21, which has an ortho-nitro group, was
been found to be more active in comparison to ebselen and
Figure 1. Molecular structure of compounds 20a (top) and 20b (bottom)
(50% ellipsoid). Selected bond lengths [ꢂ] and bond angles [8] are only
the other derivatives, which have a substituent at the 6-posi-
tion. The high catalytic activity of 21 could be attributed to
the sterically less bulky nature of the nitro group in compar-
ison to the sterically more hindered amide and oxazoline
groups.
ꢀ
ꢀ
given for 20a: C1 Se 1.940(3), Se Se# 2.328(1), Se···O1 2.729(20), C1-
Se-Se# 102.89(10), C1-Se-Se#-C1# 97.42(16), O1···Se-Se# 177.16(4).
ꢀ
Shi et al. noted that ebselen is detrimental to the biologi-
cal system at lower GSH concentration due to the undesired
reactions.[49] Recently, Jacob and co-workers described sele-
nium and tellurium compounds as redox modulators with se-
lective cytotoxicity.[50] It has been described that selenium-
and tellurium-based redox systems turn the oxidizing envi-
ronment present in certain cancer cells into a lethal cocktail
of reactive species, which forces these cells over a critical
redox threshold and finally destroys them through apoptosis.
Therefore, it is noteworthy to mention that compounds 34
and 42 or their derivatives tethered with quinine redox sys-
tems might be detrimental to the cells under oxidative stress
(certain cancer cells) by interfering with the GSSG/GSH
redox system and other thiol-containing proteins.
intramolecular Se···O1 and the intermolecular O···H N in-
teraction in 20a create hexagonal cavities in two dimensions
(see the Supporting Information, Figure S98).
Molecular structures of 34: The coordination geometry
around the selenium atom in 34 is distorted T-shaped with a
C1-Se-N1A angle of 84.26(6)8 (Figure 2).[23,24] There is a
short intramolecular distance between Se and O1B
(2.427(1) ꢂ), which suggests a strong attractive interaction
between these atoms. The molecular structure of the acid
analogue 61 (see the Supporting Information, Figure S101)
is quite similar to the structure of compound 34.
X-ray crystallographic study
Molecular structure of 20: Compounds 20a and 20b
(Figure 1) crystallize in a monoclinic crystal system with the
chiral space group C2 with an absolute structure parameter
or Flack parameter of 0.010(13) and ꢀ0.007(12) for 20a and
20b, respectively, which confirms the enantiomeric purity of
the crystals. The geometry around the selenium center in
both of the enantiomers is similar and T-shaped for each se-
lenium bonded to other selenium or carbon atoms, and in-
tramolecularly coordinated with one of the oxygen atoms
Figure 2. Molecular structure of compound 34 (50% ellipsoid). Selected
ꢀ
ꢀ
bond lengths [ꢂ] and bond angles [8]: Se C1 1.861(1), Se N1A 1.903(1),
ꢀ
(Se···O1). The dihedral angles around the Se Se bond (C1-
ꢀ
Se O1B 2.427(1), C1-Se-N1A 84.26(6), C1-Se-O1B 74.91(5), N1A-Se-
Se-Se#-C1#) are 97.42(16) (20a) and ꢀ97.37(14)8 (20b). The
O1B 159.10(5).
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12749

H. B. Singh et al.
Molecular structure of 46: The asymmetric unit of 46 com-
prises one molecule of the solvent CHCl3 (Figure 3). The
CHCl3 present in the asymmetric unit is disordered. The ge-
ometry around the selenium is T-shaped, similar to that of
compounds 20a/20b and 48 (see the Supporting Informa-
tion, Figure S100) with each selenium boded to other seleni-
um or carbon atoms, and intramolecularly coordinated with
lenenyl sulfide and diaryl dislenide intermediates obtained
from intramolecularly stabilized ebselen analogues undergo
peroxide-mediated intramolecular cyclization reactions. Cat-
alysts 34 and 42 require high concentrations of GSH to over-
come the intramolecular cyclization at the selenenic acid
stage. Therefore, one must consider these facts while consid-
ering the biological applications of such derivatives. The dis-
elenides with amino acid functionalities as intramolecularly
coordinating groups displayed much better catalytic efficien-
cies. Compound 20a and 20b displayed the best catalytic
performance among the series.
one of the oxygen (Se1···O1A, 2.665(2) ꢂ). The dihedral
[38]
ꢀ
angle around the Se1 Se2 bond (87.54(9)8) is cissoid. The
CHCl3 molecule sits in the hexagonal cavity created by the
ꢀ
intramolecular Se···O and the intermolecular O···H N inter-
action (see the Supporting Information, Figure S99). It is in-
teresting to note that compounds 20a and 46 form similar
hexagonal cavity. This could be of general structural features
for this kind of diselenides. These cavities are suitably used
to trap the CHCl3 molecule in compound 46.
Experimental Section
General experimental procedures: All organoselenium compounds were
synthesized under nitrogen or argon atmosphere by using standard
vacuum techniques. Solvents were purified by standard procedures and
were freshly distilled prior to use. 1H (400 MHz) and 13C (100.56 MHz)
NMR spectra were obtained on a Varian VS400 NMR/Bruker AV-400
NMR spectrometer and 77Se (57.26/76.31 MHz) NMR spectra were re-
corded on a Varian 300 NMR/Bruker AV-400 spectrometer. Electrospray
ionization mass spectrometry (ESI-MS) experiments were performed on
mass spectrometer equipped with a Q-Tof analyzer. In the case of isotop-
ic patterns, the m/z values are given for the most intense peak. FTIR
spectra were recorded on a Perkin–Elmer Spectrum One FTIR spectrom-
eter with KBr pellets. The kinetic experiments were carried out on a
JASCO V-570, UV-Visible spectrophotometer. All amino acids that were
used were l enantiomers if not mentioned otherwise. C-protected amino
acids were prepared by standard procedure.[51] Melting points were re-
corded on VEEGO melting point apparatus and were uncorrected. The
optical rotations were recorded on a Rudolph Autopol automatic polar-
imeter.
Figure 3. Molecular structure of compound 46 (50% ellipsoid). Selected
bond lengths [ꢂ] and bond angles [8]; chloroform has been removed for
Synthesis of 20 (modified procedure): Triethylamine (0.58 mL, 4.2 mmol)
was added to a stirred solution of diselenide 45 (0.83 g, 2.1 mmol), l-ala-
nine methyl ester hydrochloride/d-alanine methyl ester hydrochloride
ꢀ
ꢀ
ꢀ
clarity; Se1 C1A 1.938(2), Se1 Se2 2.329(1), Se2 C1B 1.933(2),
Se1···O1A 2.665(2), C1A-Se1-Se2 103.46(6), C1B-Se2-Se1 102.89(6),
C1A-Se1-Se2-C1B 87.54(9).
(0.58 g, 4.2 mmol), and HOBt (0.57 g, 4.2 mmol) in THFACTHNUTRGENUG(N 15 mL). THF
(15 mL) that contains DCC (0.93 g, 4.5 mmol) was added to the above re-
action mixture at 08C. After the addition, the ice bath was removed and
the reaction was stirred at room temperature for 8 h. The obtained pre-
cipitate was filtered. The residue, which was obtained after evaporation
of solvent, was dissolved in ethyl acetate and washed with HCl (1n), an
aqueous solution of NaHCO3 (5%), water, and brine. The obtained pre-
cipitate recrystallized from a dichloromethane/petroleum ether mixture
(3:1). Yield: 0.36 g (30%). Characterization data for 20a (l,l-alanine
methyl ester): m.p. 179–1808C (reported: 174–1758C[17]); [a]D22 =ꢀ5.16
(c=1 in CH2Cl2); [a]2D2 =ꢀ80.68 (c=1 in MeOH); 1H NMR (400 MHz,
CDCl3): d=7.87 (dd, J=8.0, 0.8 Hz, 2H), 7.59 (dd, J=8.0, 1.6 Hz, 2H),
7.32–7.22 (m, 4H), 6.89 (d, J=6.4 Hz, 2H), 4.85 (p, J=7.2 Hz, 2H), 3.82
(s, 6H), 1.57 ppm (d, J=7.2 Hz, 6H); 13C NMR (100.56 MHz, CDCl3):
d=173.6, 167.7, 133.4, 132.4, 132.2, 131.5, 127.2, 126.3, 52.9, 48.8,
18.8 ppm; 77Se NMR (57.26 MHz, CDCl3): d=450 ppm; FTIR (KBr): n˜ =
3312, 1747, 1621, 1582, 1537, 1453, 1341, 1241, 1173, 1055, 871,
747 cmꢀ1 cmꢀ1; elemental analysis calcd (%) for C22H24N2O6Se2 (570.36):
C 46.33, H 4.24, N 4.91; found: C 46.27, H 3.89, N 5.00. Characterization
data for 20b (d,d-alanine methyl ester): m.p. 179–1808C; [a]D22 = + 5.12
(c=1, CH2Cl2); [a]2D2 = +83.44 (c=1 in MeOH); 1H NMR (400 MHz,
CDCl3): d=7.87 (dd, J=8.0 0.8 Hz, 2H), 7.59 (dd, J=8.0 1.6 Hz, 2H),
7.32–7.22 (m, 4H), 6.89 (d, J=6.4 Hz), 4.85 (p, J=7.2 Hz, 2H), 3.82 (s,
6H), 1.57 ppm (d, J=7.2 Hz, 6H); 13C NMR (100.56 MHz, CDCl3): d=
173.6, 167.7, 133.4, 132.3, 132.2, 131.4, 127.1, 126.3, 52.9, 48.8, 18.8 ppm;
77Se NMR (57.26 MHz, CDCl3): d=450 ppm; FTIR (KBr): n˜ =3312,
Conclusion
It has been demonstrated that the polar and sterically bulky
amino acid substituents present at the 2,6-positions of aryl
bromides do not facilitate the SNAr displacement of Br with
Li2Se2/Na2Se2. For the first time, the DCC coupling strategy
has been used for the convenient synthesis of ebselen ana-
logues with amino acid functions as the intramolecularly co-
ordinating group. The diselenides with 2,6-carboxyl (38)/
NHS-ester (41) substitution undergo facile intramolecular
cyclization with C-protected amino acids. However, the dise-
lenides with one ortho-carboxyl (45)/NHS-ester (48) group
do not undergo any intramolecular cyclization. Although
ꢀ
2,6-disubstitution can be used for the facile synthesis of Se
N heterocyclic organoselenium compounds, the intramolecu-
lar cyclization reduces the yield of the products in the DCC
coupling reaction through side reactions. Therefore, the re-
ductive cleavage of the diselenide bond before the coupling
reactions occurs, is necessary. The ebselen analogues with in-
tramolecular coordination showed slightly higher Vmax
values than ebselen. In contrast they displayed poor catalyt-
ic activity at low GSH concentrations. It appears that the se-
1747, 1622, 1582, 1540, 1462, 1341, 1241, 1173, 1055, 871, 746 cmꢀ1 cmꢀ1
;
elemental analysis calcd (%) for C22H24N2O6Se2 (570.36): C 46.33, H 4.24,
N 4.91; found: C 46.66, H 4.10, N 5.33.
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Synthesis and Enzymatic Activity of Ebselen Analogues and Diaryl Diselenides
FULL PAPER
Synthesis of 31: Thionyl chloride (12.0 mL, 165 mmol) was added to 2-
bromo-5-tert-butyl-isophthalic acid (30)[34] (10.0 g, 33.2 mmol) in a three-
necked round bottomed flask. The reaction mixture was heated to reflux
for 3 h. Excess thionyl chloride was removed under vacuum. The ob-
tained residue was dissolved in dichloromethane (20 mL) and used for
further reaction. Triethylamine (18.5 mL, 133 mmol) and the acid chlo-
ride were added simultaneously to a stirred solution of glycine methyl
ester hydrochloride (8.3 g, 66 mmol) in dichloromethane (40 mL). The re-
action mixture was stirred overnight under a N2 atmosphere. The reaction
mixture was poured into water, extracted with dichloromethane, washed
with HCl (1n), an aqueous solution of NaHCO3 (10%), and brine. The
organic layer was dried over sodium sulfate and the solvent was evapo-
rated in vacuo to give a colorless oil, which recrystallized from dichloro-
methane and n-hexane to give a colorless solid. Yield: 10.1 g (69%); m.p.
144–1468C; 1H NMR (400 MHz, CDCl3): d=7.55 (s, 2H), 6.47 (brt, J=
4.8 Hz, 2H), 4.27 (d, J=5.2 Hz, 4H), 3.82 (s, 6H), 1.32 ppm (s, 9H);
13C NMR (100.56 MHz, CDCl3): d=170.0, 168.1, 151.8, 138.7, 127.9,
113.0, 52.7, 41.9, 35.0, 31.1 ppm; FTIR (KBr): n˜ =3295, 3068, 2955, 2923,
2852, 1758, 1647, 1585, 1548, 1312, 1266, 1208, 1013, 892, 705 cmꢀ1; ESI-
MS: m/z (%) calcd for C18H23BrN2O6: 442; found: 465 [M+Na]+ (24),
443 [M+H]+ (100), 354 [MꢀNHCH2COOMe]+ (62) cmꢀ1; elemental
analysis calcd (%) for C18H23BrN2O6 (443.29): C 48.77, H 5.23, N 6.32;
found: C 48.01, H 4.54, N 6.94.
to give a yellowish oil, which gave a pale yellow solid upon addition of
petroleum ether. A spectroscopically pure product was obtained by
column chromatography by using petroleum ether and ethyl acetate
(6%) as eluent. Yield: 0.18 g (61%); m.p. 190–1928C; 1H NMR
(400 MHz, CDCl3): d=8.24 (d, J=1.6 Hz, 1H), 7.93 (d, J=1.6 Hz, 1H),
7.55 (brt, J=5.6 Hz, 1H), 4.57 (s, 2H), 4.20 (d, J=5.2 Hz, 2H), 3.82 (s,
3H), 3.79 (s, 3H), 1.42 ppm (s, 9H); 13C NMR (100.56 MHz, CDCl3): d=
170.8, 170.1, 167.2, 166.7, 150.8, 138.9, 128.9, 128.4, 125.9, 124.2, 52.8,
52.6, 44.8, 41.8, 35.3, 31.6 ppm; 77Se NMR (57.26 MHz, CDCl3): d=
897 ppm; FTIR (KBr): n˜ =3362, 3058, 2966, 2852, 1743, 1634, 1620, 1589,
1216, 1182, 1117, 694, 583 cmꢀ1
; ESI-MS: m/z (%) calcd for
C18H22N2O6Se: 442; found: 443 [M+H]+ (100); elemental analysis calcd
(%) for C18H22N2O6Se (441.34): C 48.99, H 5.02, N 6.35; found: C 50.02,
H 5.00, N 6.74.
Method IIIa (modified method III): Triethylamine (0.29 mL, 2.1 mmol)
was added to active ester 41 (0.50 g, 0.51 mmol) and glycine methyl ester
hydrochloride (0.26 g, 2.1 mmol) in THF (15 mL) at 08C. The ice bath
was removed and the reaction mixture was stirred for additional 16 h.
The reaction mixture was poured into water, extracted with dichlorome-
thane (2ꢁ25 mL), and washed with HCl (1n), an aqueous solution of
NaHCO3 (10%), and water. The dried organic layer was evaporated to
give a colorless solid, which was recrystallized from chloroform/petrole-
um ether mixture (1:3). Yield: 0.18 g (41%)
Synthesis of 32: Compound 32 was synthesized from 30 (2.7 g, 9.0 mmol),
thionyl chloride (2.5 mL, 34 mmol), dichloromethane (25 mL), triethyl-
ACHTUNGTRENNUNGamine (5.2 mL, 37 mmol), and l-tryptophan methyl ester hydrochloride
Method IV: Thionyl chloride (5 mL) was added to diselenide 38 (0.50 g,
0.83 mmol) in a three-necked round bottomed flask. The reaction mix-
ture was heated to reflux for 6 h. Excess thionyl chloride was removed in
vacuum. The obtained residue was dissolved in CHCl3 (15 mL). Triethyla-
mine (1.0 mL, 7.2 mmol) and the acid chloride were added simultaneous-
ly to a stirred solution of glycine methyl ester hydrochloride (0.42 g,
3.3 mmol) in dry dichloromethane (10 mL) in a round bottomed flask at
08C. The reaction mixture was stirred for 16 h. Then the reaction mixture
was poured into water and extracted with dichloromethane. The organic
layer was washed with HCl (1n), an aqueous solution of NaHCO3
(10%), and brine. The organic layer was dried over sodium sulfate and
evaporated in vacuo to give a colorless solid, which was recrystallized
from CHCl3/ethyl acetate (0.5:1). Yield: 0.52 g (71%).
(4.6 g, 18 mmol), by following the procedure used for the synthesis of
compound 31. Yield: 5.5 g (87%); m.p. 149–1508C; [a]2D2 = +76.16 (c=1
in CHCl3); 1H NMR (400 MHz, CDCl3): d=10.92 (d, J=2 Hz, 2H), 8.97
(d, J=7.6 Hz, 2H), 7.54 (d, J=7.6 Hz, 2H) 7.34 (d, J=8.4 Hz, 2H), 7.21
(d, J=2 Hz, 2H), 7.11 (s, 2H), 7.10 (t, J=7.2 Hz, 2H), 6.99 (t, J=7.2 Hz,
2H), 4.66 (dt, J=9.2, 5.6 Hz, 2H), 3.44 (s, 6H), 3.21 (m, 4H), 1.21 ppm
(s, 9H); 13C NMR (100.56 MHz, CDCl3): d=172.1, 167.9, 151.6, 138.6,
136.3, 127.6, 123.5, 122.21, 119.7, 118.5, 113.1, 111.5, 109.4, 53.5, 52.7,
34.8, 31.0, 27.6 ppm; FTIR (KBr): n˜ =3419, 3401, 2956, 2923, 1733, 1652,
1518, 1457, 1437, 1225, 1021, 744 cmꢀ1; elemental analysis calcd (%) for
C36H37BrN4O6 (701.61): C 61.63, H 5.32, N 7.99; found: C 61.10, H 5.22,
N 8.14.
Synthesis of 35: n-Butyl lithium (1.5 mL, 2.4 mmol) was added to a
stirred solution of selenium (0.18 g, 2.3 mmol) in dry and degassed THF
(25 mL). The reaction mixture was stirred at room temperature for
30 min. A degassed solution of compound 31 (1.0 g, 2.3 mmol) in THF
(5 mL) was added dropwise at 08C and the reaction mixture was stirred
for further 3 h in an ice bath. The reaction mixture was poured into
water and extracted with dichloromethane (2ꢁ25 mL). The organic
layers were combined, dried over sodium sulfate, and evaporated to give
a colorless oil, which was recrystallized from dichloromethane/n-hexane
Synthesis of 34
Method I: THF (25 mL) was added to selenium powder (0.54 g,
6.8 mmol), freshly cut lithium (0.05 g, 7.1 mmol), and a catalytic amount
of naphthalene in a three-necked round-bottomed flask equipped with a
reflux condenser. After 6 h of heating to reflux, the reaction mixture was
brought down to room temperature and the slurry of Li2Se2 was allowed
to settle down. THF was removed by using a syringe. A solution of com-
pound 31 (2.0 g, 4.5 mmol) in DMF (20 mL) was added to the residue at
0–58C. The reaction mixture was stirred overnight and then poured into
water (50 mL). The aqueous layer was extracted with dichloromethane
(2ꢁ50 mL). The organic layers were combined, dried with sodium sul-
fate, and evaporated to give a orange oil. Purification by column chroma-
tography by using silica gel as stationary phase and dichloromethane/
methanol (1:0.01) as mobile phase gave compound 34. Yield: 0.14 g
(7%).
(4:1) to give
a colorless solid. Yield: 0.31 g (27%); m.p. 70–728C;
1H NMR (400 MHz, CDCl3): d=7.63 (s, 2H), 6.89 (brt, J=4.8 Hz, 2H),
4.29 (d, J=5.2 Hz, 4H), 3.8 (s, 3H), 2.86 (t, J=7.6 Hz, 2H), 1.52 (p, J=
7.6 Hz, 2H), 1.32 (m, 11H), 0.87 ppm (t, J=7.2 Hz, 3H); 13C NMR
(100.56 MHz, CDCl3): d=170.2, 169.4, 152.5, 142.4, 127.3, 120.3, 52.7,
42.0, 35.1, 32.3, 31.3, 31.2, 23.0, 13.7 ppm; 77Se NMR (57.26 MHz,
CDCl3): d=234 ppm; FTIR (KBr): n˜ =3278, 3071, 2956, 2868, 1758, 1651,
1543, 1369, 1305, 1262, 1203, 1177, 1014, 895, 700, 562 cmꢀ1; ESI-MS: m/z
(%) calcd for C22H32N2O6Se: 500; found: 501 [M+H]+ (100); elemental
analysis calcd (%) for C22H32N2O6Se (499.46): C 52.90, 6.46, 5.61; found:
C 51.92, H 6.01, N 5.99.
Method II: Bromine (0.05 mL, 1.0 mmol) in CHCl3 (5 mL) was added to
a solution of monoselenide 35 (0.12 g, 0.3 mmol) in CHCl3 (10 mL) at
08C. The reaction mixture was stirred for 6 h at room temperature. The
reaction mixture was poured into water and was extracted with CHCl3
(20 mL). After evaporation compound 34 was obtained as a colorless
solid in nearly quantitative yields.
Synthesis of 36: Compound 36 was synthesized by using the procedure
adopted for the synthesis of 35 by using selenium (0.36 g, 4.6 mmol) in n-
butyllithium (3.0 mL, 4.8 mmol) and compound 32 (1.6 g, 2.3 mmol). The
obtained residue was recrystallized from methanol/water (10:1) to give a
colorless solid. Yield: 0.47 g (27%); m.p. 133–1358C; [a]2D2 = +55.84 (c=
1 in CHCl3); 1H NMR (400 MHz, CDCl3): d=8.17 (brs, 2H), 7.6 (d, J=
7.2 Hz, 2H), 7.45 (s, 2H), 7.30 (d, J=8.0 Hz, 2H), 7.17 (t, J=6.8 Hz,
2H), 7.08 (m, 4H), 6.86 (d, J=7.2 Hz, 2H), 5.10 (q, J=5.6 Hz, 2H), 3.73
(s, 6H), 3.5–3.6 (dq, J=14.8, 5.6 Hz, 4H), 2.6 (m, 2H), 1.1–1.29 (m, 13H)
0.73 ppm (t, J=7.2 Hz, 3H); 13C NMR (100.56 MHz, CDCl3): d=172.4,
169.0, 152.1, 142.3, 136.3, 127.7, 127.2, 123.3, 122.4, 120.6, 119.9, 118.8,
111.4, 110.1, 53.7, 52.6, 34.9, 32.1, 31.1, 27.9, 22.9, 13.7 ppm; 77Se NMR
Method III: Triethylamine (0.20 mL, 1.4 mmol) was added to a stirred so-
lution of diselenide 38 (0.20 g, 0.33 mmol), glycine methyl hydrochloride
(0.17 g, 1.4 mmol), and HOBt (0.20 g, 1.5 mmol) in THF (20 mL) at ice-
cold condition. DCC (0.33 g, 1.6 mmol) in THF (5 mL) was added drop-
wise to the reaction mixture. The reaction mixture was stirred at RT over-
night. The resultant reaction mixture was poured into water and extract-
ed with dichloromethane (2ꢁ40 mL). The organic layers were combined
and washed with HCl (1n), an aqueous solution of NaHCO3 (10%), and
water. The organic layer was dried over sodium sulfate and evaporated
Chem. Eur. J. 2011, 17, 12741 – 12755
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12751

H. B. Singh et al.
(57.26 MHz, CDCl3): d=233 ppm; FTIR (KBr): n˜ =3420, 3407, 2952,
2599, 1734, 1646, 1514, 1459, 1432 1224, 1010, 743, 615; ESI-MS: m/z (%)
calcd for C40H46N4O6Se: 758; found: 760 [M+H]+ (100); elemental analy-
sis calcd (%) for C40H46N4O6Se (757.78): C 63.40, H 6.12, N 7.39; found:
C 63.73, H 5.89, N 8.02.
[a]2D2 =ꢀ41.96 (c=1 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=8.36 (s,
1H), 8.20 (d, J=1.6 Hz, 1H), 8.10 (s, 1H), 7.68 (dd, J=8.4, 0.8 Hz, 1H),
7.51–7.04 (m, 10H), 5.65 (dd, J=8.4, 6.4 Hz, 1H), 5.14 (dt, J=8.0, 5.2 Hz,
1H), 3.75 (s, 3H), 3.66 (s, 3H), 3.58 (dq, J=15.2, 6.4 Hz, 2H) , 3.40 (m,
2H), 1.23 ppm (s, 9H); 13C NMR (100.56 MHz, CDCl3): d=172.0, 171.9,
167.1, 166.4, 150.7, 139.8, 136.3, 136.2, 129.4, 129.0, 127.7, 127.5, 125.2,
124.7, 123.3, 122.9, 122.5, 122.1, 120.3, 119.6, 118.7, 118.1, 111.8, 111.3,
110.5, 109.3, 56.6, 54.4, 52.8, 52.6, 35.0, 31.5, 29.1, 27.5 ppm; 77Se NMR
(57.26 MHz, CDCl3) d=876 ppm; FTIR (KBr): n˜ =3414, 2954, 1740,
1618, 1530, 1439, 1366, 1221, 1098, 743, 587 cmꢀ1; ESI-MS: m/z (%) calcd
for C36H36N4O6Se: 700; found: 740 [M+K]+ (2), 723 [M+Na]+ (12), 701
[M+H]+ (100); elemental analysis calcd (%) for C36H36N4O6Se (699.65):
C 61.80, H 5.19, N 8.01; found: C 60.66, H 4.82, N 7.70.
Synthesis of 39
Method III: The synthesis of ebselen 39 was attempted by following
adopting method III for the synthesis of compound 34 by using diselenide
38 (0.31 g, 0.5 mmol), l-phenylalanine methyl ester hydrochloride (0.43 g,
2 mmol), DCC (0.43 g, 2.1 mmol), and HOBt (0.28 g, 2.1 mmol). The
crude mixture, which was obtained after the workup, was purified by
column chromatography on a silica gel column (60–120 mesh) as station-
ary phase and by using ethyl acetate/petroleum ether (1:3) as mobile
phase. Compound 39: yield: 0.06 g (10%); m.p. 84–878C; [a]2D2 =ꢀ13.36
(c=1 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=8.22 (d, J=1.2 Hz,
1H), 7.71 (d, J=1.4 Hz, 1H), 7.34–7.12 (m, 10H) 7.10 (d, J=7.6 Hz,
1H), 5.58 (q, J=6.4 Hz, 1H), 5.10 (dt, J=7.2, 4.8 Hz, 1H), 3.79 (s, 3H),
3.68 (s, 3H), 3.45 (dd, J=14.0, 6.4 Hz, 1H), 3.29 (dd, J=5.6, 1.2 Hz, 2H),
3.23 (dd, J=14.4, 8.8 Hz, 1H), 1.34 ppm (s, 9H); 13C NMR (100.56 MHz,
CDCl3) d=171.8, 171.6, 166.9, 166.3, 150.7, 139.7, 136.4, 135.6, 129.4,
129.2, 128.9, 128.6, 127.6, 127.0, 125.1, 124.7, 57.4, 54.1, 52.8, 52.6, 39.0,
37.9, 35.1, 31.5 ppm; 77Se NMR (57.26 MHz, CDCl3): d=877 ppm; FTIR
(KBr): n˜ =3245, 3064, 3029, 2954, 1746, 1615, 1547, 1366, 1292, 1110, 742,
700 cmꢀ1; ESI-MS m/z calcd for C32H34N2O6Se: 622; found: 623 [M+H]+
(100); elemental analysis calcd (%) for C32H34N2O6Se (621.58): C 61.83,
H 5.51, N 4.51; found: C 61.72, H 5.42, N 4.90. Compound 40: yield:
Synthesis of 45 (modified procedure): Dry THF (50 mL) was added to a
mixture of selenium (2.0 g, 25 mmol), sodium (0.60 g, 26 mmol), and
naphthalene (0.50 g, 4.0 mmol). The reaction mixture was heated to
reflux for 6 h and then brought to room temperature. The dark brown
slurry of Na2Se2 was allowed to settle down. The supernatant liquid was
removed by using a syringe under a nitrogen atmosphere. The precipitate
was dissolved in water (30 mL) to give a dark brown solution.[52] Sodium
hydroxide (1.0 g, 25 mmol) was added to the above-described solution.
The obtained disodium diselenide was added dropwise to a stirred solu-
tion of 2-carboxybenzenediazonium chloride, which was prepared in ad-
vance by the reaction of anthranilic acid (3.0 g, 22 mmol), sodium nitrite
(1.8, 26 mmol), and concentrated HCl (4.5 mL) in water (30 mL) at 0–
58C. After complete addition of Na2Se2, the pH value of the reaction
mixture was adjusted to basic (by using litmus paper) by adding sodium
hydroxide. The reaction mixture was stirred at room temperature for ad-
ditional 2 h. The reaction mixture was acidified with concentrated HCl to
give a brown precipitate. The precipitate was purified by recrystallization
from methanol. Yield: 3.3 g (74%); 1H NMR (400 MHz, [D6]DMSO]):
d=13.77 (brs, 2H), 8.03 (dd, J=7.6, 1.2 Hz, 2H), 7.67 (d, J=8.4 Hz,
2H), 7.51 (dt, J=8.4, 1.2 Hz, 2H), 7.36 ppm (t, J=7.2 Hz, 2H);
77Se NMR (57.26 MHz, CD3OD): d=441 ppm.
1
0.20 g (31%); m.p. 180–1818C; [a]2D2 = +93.84 (c=1 in CH2Cl2); H NMR
(400 MHz, CDCl3): d=8.51 (d, J=2.0 Hz, 1H), 7.49 (d, J=2.4 Hz, 1H),
7.26–7.36 (m, 3H), 7.16 (dd, J=8.0, 2.0 Hz, 2H), 6.54 (d, J=7.6 Hz, 1H),
5.07 (dt, J=7.2, 4.8 Hz, 1H), 4.85 (tt, J=12.0, 3.2 Hz, 1H), 3.83 (s, 3H),
3.63 (m, 1H), 3.34 (dq, J=13.6, 5.6 Hz, 2H), 2.40 (q, J=12.8 Hz, 2H),
1.82–1.26 ppm (m, 29H); 13C NMR (100.56 MHz, CDCl3): d=171.9,
167.3, 164.4, 149.2, 140.1, 135.7, 132.5, 131.0, 130.2, 129.5, 128.9, 128.7,
127.8, 127.6, 60.8, 60.3, 53.7, 52.8, 37.7, 34.8, 33.8, 33.8, 31.2, 30.2, 29.7,
26.8, 26.7 26.0, 25.9, 24.3, 24.2 ppm; 77Se NMR (57.26 MHz, CDCl3): d=
384 ppm; FTIR (KBr): n˜ =3303, 2932, 2853, 1747, 1649, 1610, 1536, 1447,
Synthesis of 46: Compound 46 was synthesized, from diselenide 45
(0.50 g, 1.3 mmol), glycine methyl ester hydrochloride (0.33 g, 2.6 mmol),
HOBt (0.35 g, 2.6 mmol), triethylamine (0.36 mL, 2.6 mmol), and DCC
(0.56, 2.7 mmol) by using the procedure adopted for the synthesis of 20a/
20b. Yield: 0.38 g (56%); m.p. 190–1918C; 1H NMR (400 MHz, CDCl3):
d=7.88 (d, J=7.6 Hz, 2H), 7.59 (d, J=6.4 Hz, 2H), 7.37–7.20 (m, 4H),
6.76 (brt, 2H), 4.29 (d, J=5.2 Hz, 4H), 3.83 ppm (s, 6H); 13C NMR
(100.56 MHz, CDCl3): d=170.5, 168.3, 133.6, 132.3, 131.7, 127.2, 126.4,
52.8, 42.0 ppm; 77Se NMR (57.26 MHz, CDCl3): d=448 ppm; FTIR
(KBr): n˜ =3423, 3314, 2940, 1754, 1632, 1536, 1405, 1370, 1210, 1004,
742 cmꢀ1; elemental analysis calcd (%) for C20H20N2O6Se2 (722.55): C
44.30, H 3.72, N 5.17; found: C 44.10, H 3.43, N 5.37.
1366, 1207, 1107, 890, 702, 623 cmꢀ1
; ESI-MS: m/z (%) calcd for
C35H45N3O4Se: 651; found: 652 [M+H]+ (100); elemental analysis calcd
(%) for C35H45N3O4Se (650.71): C 64.60, H 6.97, N 6.46; found: C 64.39,
H 6.63, N 6.29.
Method IIIa: Compound 39 was synthesized from active ester 41 (0.60 g.
0.61 mmol), l-phenylalanine methyl ester hydrochloride (0.52 g,
2.4 mmol), and triethylamine (0.34 mL, 2.4 mmol) by adopting method
IIIa, which was described for the synthesis of compound 34. The obtained
residue was purified by column chromatography by using petroleum
ether/ethyl acetate (20%). Yield: 0.31 g (42%).
Synthesis of 47: Compound 47 was synthesized from diselenide 45
(0.80 g, 2.0 mmol), l-phenylalanine methyl ester hydrochloride (0.86 g,
4.0 mmol), HOBt (0.57 g, 4.2 mmol), triethylamine (0.56 mL, 4.0 mmol),
and DCC (0.86 g, 4.2 mmol) by using the procedure described for the
synthesis of compound 46. Yield 0.48 g (33%); m.p. 173–1758C; [a]2D2 = +
139.88 (c=1 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=7.86 (dd, J=
8.0, 0.8, 2H), 7.41 (dd, J=7.6, 1.6 Hz, 2H), 7.15–7.33 (m, 14H), 6.68 (d,
J=7.6 Hz, 2H), 5.12 (dt, 7.6, 5.6 Hz, 2H), 3.79 (s, 6H), 3.28 ppm (dq, J=
14.0, 6.0 Hz, 4H); 13C NMR (100.56 MHz, CDCl3): d=172.0, 167.7, 135.9,
133.5, 132.5, 132.3, 131.5, 129.6, 128.9, 127.5, 127.1, 126.3, 53.9, 52.7,
37.9 ppm; 77Se NMR (57.26 MHz, CDCl3): d=448 ppm; FTIR (KBr): n˜ =
3291, 2927, 1739, 1633, 1537, 1436, 1283, 1025, 746, 701 cmꢀ1; ESI-MS: m/
z (%) calcd for C34H32N2O6Se2: 724; found: 725 [M+H]+ (100); elemen-
tal analysis calcd (%) for C34H32N2O6Se2 (722.55): C 56.52, H 4.46, N
3.88; found C 56.95, H 4.22, N 4.48.
Synthesis of 42: Compound 42 was synthesized from active ester 41
(0.60 g, 0.61 mmol), l-alanine methyl ester hydrochloride (0.34 g,
2.4 mmol), and triethylamine (0.34 mL, 2.4 mmol), using method IIIa.
The obtained residue was purified by column chromatography by using
dichloromethane/methanol (1:0.04). Yield: 0.20 g (35%); m.p. 95–978C;
[a]2D2 = +5.72 (c=1 in CHCl3); 1H NMR (400 MHz, CDCl3): d=8.16 (d,
J=1.2 Hz, 1H), 7.99 (d, J=1.6 Hz, 1H), 7.85 (d, J=7.2 Hz, 1H), 5.22 (q,
J=7.2 Hz, 1H), 4.75 (p, J=6.8 Hz, 1H), 3.71 (s, 3H), 3.66 (s, 3H), 1.55
(d, J=7.2 Hz, 3H), 1.48 (d, J=7.2 Hz, 3H), 1.24 ppm (s, 9H); 13C NMR
(100.56 MHz, CDCl3): d=173.3, 172.6, 166.8, 166.5, 150.9, 139.6, 129.4,
128.9, 125.5, 125.0, 52.9, 52.6, 51.9, 49.2, 35.2, 31.5, 18.7, 18.2 ppm;
77Se NMR (57.26 MHz, CDCl3): d=865 ppm; FTIR (KBr): n˜ =3261,
3074, 2956, 1747, 1615, 1550, 1452, 1366, 1296, 1269, 1213, 1153, 1055,
982, 781, 589 cmꢀ1; ESI-MS: m/z (%) calcd for C20H26N2O6Se: 470;
found: 471 [M+H]+ (100); elemental analysis calcd (%) for
C20H26N2O6Se (469.39): C 51.18, H 5.58, N 5.97; found: C 51.63, H 5.54,
N 6.17.
Synthesis of 48: A solution of DCC (1.3 g, 6.2 mmol) in THF (10 mL)
was added to a stirred solution of diselenide 45 (1.2 g, 3.0 mmol) and
NHS (0.70 g, 6.0 mmol) in THF (15 mL) at 0–58C. The reaction mixture
was stirred at room temperature for 6 h. After filtration the filtrate was
diluted with dichloromethane (40 mL). The diluted solution was washed
with an aqueous solution of NaHCO3 (5%) and water. The solution was
dried over sodium sulfate and evaporated to give an off white precipitate.
Synthesis of 43: Compound 43 was synthesized from active ester 41
(0.80 g, 0.81 mmol), l-tryptophan methyl ester hydrochloride (0.82 g,
3.2 mmol), and triethylamine (0.45 mL, 3.2 mmol) by following method
IIIa. The obtained residue was purified by column chromatography by
using chloroform/methanol (1:0.01). Yield 0.44 g (39%); m.p. 143–1468C;
12752
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 12741 – 12755

Synthesis and Enzymatic Activity of Ebselen Analogues and Diaryl Diselenides
FULL PAPER
Yield 1.3 g (73%); m.p. 145–1508C (decomp); 1H NMR (400 MHz,
[D6]DMSO): d=8.32–7.58 (m, 8H), 2.98 ppm (s, 8H); 13C NMR
(100.56 MHz, [D6]DMSO): d=170.4, 162.3, 136.4, 135.3, 132.2, 130.7,
127.7, 122.6, 25.7 ppm; 77Se NMR (57.26 MHz, [D6]DMSO): d=459 ppm;
FTIR (KBr): n˜ =1739, 1454, 1234, 1204, 1069, 995, 739, 646 cmꢀ1; ESI-
MS: m/z (%) calcd for C22H16N2O8Se2: 596; found: 619 [M+Na]+ (8),
482 [MꢀNHS]+ (100); elemental analysis calcd (%) for C22H16N2O8Se2
(594.29): C 44.46, H 2.71, N, 4.71; found: C 43.57, H 2.35, N 4.71.
tion was carried out in a phosphate buffer (1.00 mL, 100 mm, pH 7.5) that
contains EDTA (1 mm), NADPH (0.23 mm), and the catalyst (20 mm).[6a]
The concentration of GSH was varied in the range 0.5–2.5 mm for 1, 34,
42, and 46, and in the range 0.025–0.5 mm for 20a and 20b. The reaction
was initiated by the addition of H2O2 (0.26 mm). The decrease in
NADPH concentration was followed at l=340 nm (emax =6.22ꢁ
103 mꢀ1 cmꢀ1) as a measure of glutathione peroxidase activity by using an
UV/Vis spectrophotometer. Each of the measurement was carried out in
triplicates. The initial reduction rate (5–10% consumption of NADPH)
was expressed as mm of NADPH depleting per minute (mmminꢀ1) after
the addition of H2O2. A plot of the inverse of initial reaction rate versus
the inverse of GSH concentration gave the catalytic parameters, such as
the maximum velocities (Vmax), the Michaelis constants (KM), the catalyt-
ic constants (kcat), and the catalytic efficiencies (h) for the reduction of
H2O2 by GSH in the presence of ebselen analogues and diselenides. The
Vmax values of the new catalysts are expressed without background cor-
rection for the reaction between H2O2 and GSH. The Vmax value of ebse-
len has been used as a reference for the comparison of the activity of the
catalysts. Similarly, the GPx-like activity and the catalytic parameters for
the diselenides 5, 20a, and 46 were obtained from a benzene thiol
assay.[6b] The rate of formation of Ph2S2 was monitored at l=305 nm
(emax =1.24ꢁ103 mꢀ1 cmꢀ1). The full experimental details are given in the
Supporting Information. The samples used for in situ 77Se NMR spectros-
copy were degassed before the experiment by using argon gas. The ex-
periment was conducted under an argon atmosphere.
Synthesis of 50: Thionyl chloride (2.5 mL, 34.0 mmol) was added to dise-
lenide 45 (1.0 g, 2.5 mmol) in a three necked round bottomed flask. The
reaction mixture was heated to reflux for 6 h. Excess thionyl chloride was
removed under vacuum. The obtained residue was dissolved in dichloro-
methane (10 mL). A solution of glycine methyl ester hydrochloride of
(0.63 g, 5.0 mmol) and triethylamine (2.1 mL, 15 mmol) in dichlorome-
thane (15 mL) was added to the reaction mixture at 08C. The ice bath
was removed and the reaction mixture was stirred for additional 3 h. The
reaction mixture was poured into water and extracted with dichlorome-
thane (2ꢁ20 mL). The organic layer was washed with HCl (1n), an aque-
ous solution of NaHCO3 (5%), and water. The organic layer was dried
over sodium sulfate and evaporated to give a dark brown solid, which
was purified by column chromatography on silica gel (100–200 mesh) by
using dichloromethane. Yield: 0.66 g (49%); m.p. 143–1458C (reported
141–1458C[16]); 1H NMR (400 MHz, CDCl3): d=8.07 (d, J=8.0 Hz, 1H),
7.67–7.60 (m, 2H), 7.46–7.42 (m, 1H), 4.62 (s, 2H), 3.8 ppm (s, 3H);
13C NMR (100.56 MHz, CDCl3): d=169.3, 167.9, 139.0, 132.6, 129.1,
126.4, 126.1, 124.2, 52.8, 45.6 ppm; 77Se NMR (57.26 MHz, CDCl3): d=
933 ppm; ESI-MS: m/z (%) calcd for C10H9NO3Se: 271; found: 272
[M+H]+ (100); FTIR (KBr): n˜ =2926, 1746, 1596, 1563, 1444, 1403, 1356,
X-ray crystallography: The single-crystal X-ray diffraction measurements
for compounds 20a, 20b, 34, 46, 48, and 61 were performed on a diffrac-
tion measurement device with graphite monochromated MoKa radiation
(l=0.7107 ꢂ). The crystal structure refinement data are given in Table 5
(20a, 20b, 34, and 46) and Table S25 in the Supporting Information (48
and 61). The structures were solved by direct methods (SHELXS-97) and
full-matrix least squares refinement on F2 (SHELXL-97).[55] Hydrogen
atoms were localized by geometrical means. A riding model was chosen
for refinement. CCDC-816991 (20a), 816992 (20b), 816993 (34), 816994
(46), 816995 (48), and 830722 (61) contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif.
1208, 733, 557 cmꢀ1
; elemental analysis calcd (%) for C10H9NO3Se
(270.14): C 44.46, H 3.36, N 5.18; found: C 44.28, H 2.94, N 4.84.
Synthesis of 51: Compound 51 was synthesized from diselenide 45 (2.1 g,
5.2 mmol), thionyl chloride (2.5 mL, 34 mmol), CHCl3 (30 mL), triethyla-
mine (4.5 mL, 32 mmol), and l-tryptophan methyl ester hydrochloride
(2.6 g, 10.4 mmol) by following the procedure used for the preparation of
compound 50. Yield 2.4 g (58%); m.p. 66–698C; [a]2D2 = +14.88 (c=1 in
CH2Cl2); 1H NMR (400 MHz, CDCl3): d=8.19 (brs, 1H), 8.03 (d, J=
7.6 Hz, 1H), 7.64 (d, J=7.4 Hz, 1H), 7.57 (m, 2H), 7.39 (m, 1H), 7.32 (d,
J=4 Hz, 1H), 7.15–7.09 (m, 2H), 7.01 (d, J=3.6 Hz, 1H), 5.72 (dt, J=
7.6, 6.8 Hz, 1H), 3.67 (s, 3H), 3.40 ppm (dq, J=14.8, 6. 4 Hz, 2H);
13C NMR (100.56 MHz, CDCl3): d=171.9, 167.9, 139.7, 136.2, 132.3,
128.7, 127.4, 126.7, 126.1, 123.9, 123.1, 122.2, 119.6, 118.5, 111.4, 109.6,
56.7, 52.7, 29.1 ppm; 77Se NMR (57.26 MHz, CDCl3): d=903 ppm; FTIR
(KBr): n˜ =3412, 3258, 3055, 2945, 2841, 1736, 1613, 1444, 1339, 1223,
1096, 739, 675 cmꢀ1; ESI-MS: m/z (%) calcd for C19H16N2O3Se: 400;
found: 401 [M+H]+ (100); elemental
analysis calcd (%) for C19H16N2O3Se
Table 5. Crystal structure refinement data for 20a/20b, 34, and 46.
(399.30):
C 57.15, H 4.04 N 7.02;
20a
20b
34
46
found: C 56.30, H 3.58, N 6.95.
Computational methods: All calcula-
tions were performed by using the
Gaussian 03 suite of quantum chemical
empirical formula
formula weight
crystal system
space group
a [ꢂ]
b [ꢂ]
c [ꢂ]
a [8]
b [8]
C22H24N2O6Se2
570.35
monoclinic
C2
12.891(3)
9.116(3)
11.010(3)
90
118.519(14)
90
1136.8(5)
2
1.666
3.294
11318
C22H24N2O6Se2
570.35
monoclinic
C2
12.9381(15)
9.1446(8)
11.0577(14)
90
118.667(16)
90
1147.9(2)
2
1.650
3.262
7009
C18H22N2O6Se
441.34
C21H21Cl3N2O6Se2
661.67
monoclinic
P21/c
9.2205(2)
18.5102(3)
15.6267(3)
90
97.507(2)
90
2644.20(9)
4
1.662
3.138
36232
triclinic
programs.[53]
The
computational
¯
P1
8.4216(4)
10.1196(4)
12.1116(5)
107.052(4)
107.691(4)
96.113(3)
918.21(7)
2
1.596
2.083
13775
0.0281
models were fully optimized by using
DFT calculations at the B3LYP/6-
31g(d) level of theory and character-
ized by frequency calculation. The
input geometries for all the models
compounds were created form the X-
ray crystal coordinates of compound
34 and 46. NBO analysis was carried
out on the optimized geometries at the
g [8]
V [ꢂ3]
Z
1calcd [Mgmꢀ3
]
absorption coefficient [mmꢀ1
reflns collected
]
B3LYP/6-311gACHTUNGTRENNUNG
(d, p) level of theory.[54]
Glutathione peroxidase activity: The
assay was carried out at room temper-
ature [(25ꢁ1)8C]. The peroxide con-
tent of the stock solution was estimat-
ed before the experiments by standard
permanganometry titrations. The reac-
final R(F) [I>2s(I)][a]
wR(F2) indices [I>2s(I)][a]
data/restraints/parameters
goodness-of-fit on F2
0.0340
0.0827
3923/1/131
1.197
0.0393
0.0808
3476/1/147
1.015
0.0330
0.0761
8879/12/339
1.074
0.0587
6044/0/249
1.005
2
2
2
[a] Definitions: R(Fo)=Sj jFo jꢀjFc j j/SjFo j and wR
(Fo )={S[w
U
G
.
Chem. Eur. J. 2011, 17, 12741 – 12755
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12753

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Acknowledgements
H.B.S. gratefully acknowledges Department of Science and Technology,
New Delhi, for the Ramanna Fellowship. We are thankful to Prof.
Udai P. Singh, IIT Roorkee, for his help in collecting the single-crystal X-
ray data for compound 20a. K.S. is thankful to CSIR, New Delhi, for
SRF and SAIF, IIT Bombay, for providing analytical facility. K.S. is in-
debted to Prof. Sandeep Verma and his former PhD student Dr. Surajit
Gosh, IIT Kanpur, for their kind help in teaching him the art of DCC
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www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 12741 – 12755

Synthesis and Enzymatic Activity of Ebselen Analogues and Diaryl Diselenides
FULL PAPER
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Received: March 26, 2011
Revised: August 4, 2011
Published online: September 28, 2011
Chem. Eur. J. 2011, 17, 12741 – 12755
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12755